FIG. 1.

Trimerization of mutant EnvAs. 293T cells were transfected with pCB6-EnvA DNA by the calcium phosphate method and induced with 10 mM sodium butyrate 24 h later. Sixteen to 18 h posttransfection, cells were harvested, lysed in 1% NP-40 buffer containing protease inhibitors (3), and subjected to sucrose density centrifugation in 10 to 30% sucrose in n-octyl glucoside buffer as described previously (3). Fractions (500 μl each) were collected and processed for Western blot analysis with the anti-Ngp37 antibody which recognizes both TM and the full-length EnvA precursor, pr95 (10). The gp37 band is shown. The gradient density increases from left to right. C9S, C45S, and C9,45S contain serines in place of the cysteine at the numbered site(s); numbering is from the initial residue of the mature TM subunit. The numbered lanes contain samples from the specified gradient fractions. B denotes a sample from the bottom of the centrifuge tube. WTA, wild-type EnvA.